HELICOBACTER PYLORI PLASMIDS--IMPACT ON STRAIN DIVERSITY

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DESCRIPTION (Adapted from the Applicant's Abstract): This project involves the microorganism Helicobacter pylori, a causal agent of gastroduodenal disease. Most early studies of H. pylori were clinical/histological observations and only recently have laboratories begun molecular investigations. Few studies mention possible biological roles of H. pylori plasmid DNA, especially in minority populations. This is the focus of the applicant's investigations. Three H. pylori plasmids,pHPM179, pHPM180, and pHPM186 are different in size but share a significant amount of identity, especially within an ORF that could encode a Rep protein for theta-type replication. Both pHPM179 and pHPM180 contain DNA sequences with some sequence identity to chromosomal pathogenicity genes: pHPM179 contains a short (44 bp) region that is identical to the junction between chromosomal DNA and the pathogenicity island (PAI) recently described; pHPM180 contains two 232 bp direct repeats that have some sequence identity with a known 102 bp sequence within the cagA gene of the PAI; pHPM179 also carries a transposon of unknown origin; and pHPM186 carries two copies of the H. pylori-specific insertion sequence, IS605 as well as 6 kb of chromosomal sequence. All of these observations lead to the strong possibility that plasmids play a significant role in H. pylori pathogenesis, either directly or as vehicles for horizontal transmission of pathogenesis genes. The long term goal is to use molecular techniques to study the role of plasmid DNA in H. pylori. These proposed studies are an extension of the above observations and provide information necessary to understand the role of H. pylori plasmid DNA in pathogenesis. Specific Aim 1: Test the hypothesis that many large H. pylori plasmids contain chromosomal DNA. Determine which chromosomal DNA is present on plasmids and whether IS605 is always present. Specific Aim 2: Investigate the extent and nature of plasmid integration and excision from the chromosome. The hypothesis is that some plasmids integrate into and excise from the chromosome, thus acquiring chromosomal sequences. Excision may lead to deletion formation in the chromosome. Specific Aim 3: Investigate the extent and nature of the pBPM179 transposon DNA and the 232 hp direct repeats. Additional occurrences of these sequences will be sought in both chromosomal and plasmid DNA. Specific Aim 4: Investigate the nature of H.pylori plasmid repA. The hypothesis is that RepA protein has toxic effects on E. coli replication. Plasmids will be constructed to allow analysis of this effect and to allow purification of RepA protein for in vitro studies.